Nucleic acid detection method using nucleotide probes enabling both specific capture and detection

ABSTRACT

A nucleic acid detection assay is disclosed which assay comprises hybridizing a large nucleic acid probe (i.e. preferably more than 100 bp long) with a RNA or DNA target nucleic acid present in a biological sample. The probe in this method is to comprise both a detection element and a capture element and is to to be degradable by a suitable enzyme when it is single stranded. Following hybridization the sample is treated with a enzyme capable of degrading unhybridized nucleic acids. Then the hybrids are captured by contacting them with a solid support comprising an element capable of binding with the capture element of the probe. Finally the detection element of the probe is used to detect the probe, thereby indicating that the target nucleic acid is present in the biological sample.

This application is a 371 of PCT/FR96/01201 filed Jul. 30, 1996.

The present invention relates to a method for detecting nucleic acidsusing nucleotide probes permitting both specific capture and detection.The present invention permits automation of the large-scale specificdetection of nucleic acids using a single-stranded ribonucleotide probecomplementary to a target nucleotide sequence, said probe permittingboth capture on a suitable surface and rapid and sensitive detection.

As a result of the major genome sequencing and analysis programmes,biology some years ago entered a new era, that of the massiveacquisition of data based on the intensive use of improved existingtechniques. In five to ten years time, the genome of several modelorganisms, as well as the human genome, will be completely sequenced.Since the number of genes encoded by the human genome is calculated atbetween 50,000 and 100,000, several thousand sequences of coding regions(cDNA) and also of regulatory regions (promoters) will have to belisted, analyzed and integrated in databases, and their expressionprofiles in some hundred tissues or cells will be able to beestablished.

The expression profile of a gene consists in studying the relativeabundance of the corresponding messenger RNA (mRNA) expressed inparticular cells (for example belonging to different tissues, orrepresenting different stages of development, particular pathologies,induction by drugs, and the like). Establishment of the expressionprofiles of genes hence requires the specific and sensitive detection ofmRNAs.

The detection of specific mRNAs may be undertaken by two different typesof approach: those based on amplification techniques and those based onhybridization techniques.

The use of amplification techniques such as RT-PCR (reversetranscriptase-polymerase chain reaction) appears to be of limitedapplicability in a planned large-scale study of mRNA expression profileson account of the cost which would be entailed in synthesizing the largenumber of oligonucleotides needed for carrying it out (for 50,000different genes, the minimum number would be 100,000 oligonucleotides).

The “sandwich” hybridization technique described for the first time byDunn A. R. and Hassell J. A. (1977, cell, 12, 23-36) was developed inorder to avoid the purification and immobilization of target nucleicacids which formerly necessitated detection after solid-phasehybridization. This technique is based on the existence of twononoverlapping probes directed against the same target nucleic acid. Thefirst probe is immobilized on a solid surface and permits capture of thetarget. The second probe possesses a tracer in its sequence and permitsdetection of the target. This technique is essentially used for thedetection of amplification products. In actual fact, the probes used aresmall (from 20 to 25 nucleotides) and, unlike long probes, they do notenable strong hybridization signals to be obtained. This technique isunsuitable for the simultaneous detection of a large number of targets,since each probe necessitates hybridization temperatures which can bedifferent.

The second approach is based on hybridization techniques that do notinvolve amplification, and which hence require great sensitivity. Thereare several formats which may be used to detect a specifichybridization: liquid-phase hybridization; solid-phase hybridization; insitu hybridization on tissues or cell bodies. In all cases, the probe isa nucleic acid (DNA or RNA) capable of hybridizing with a complementarynucleic acid sequence: the target (DNA or RNA). The kinetics of thedifferent nucleic acid hybridization reactions are well known (Brittenet al., 1974, Methods Enzymol, 29, p. 363; Kohme et al., 1977Biochemistry, 16 pp. 5329-5341), and knowledge of the parametersinvolved in the rate of hybridization as well as in the stability of thetarget-probe hybrids enables the optimal conditions to be determined forobtaining the best signal-to-background ratio.

Liquid-phase hybridization is very efficient and has the advantage ofaffording the fastest rate of hybridization. However, it is difficult toseparate the free probe from the hybridized probe. Solid-phasehybridization methods which involved immobilization of the target (or ofthe probe) on a solid surface (e.g. nitrocellulose, nylon) are easier tocarry out from the standpoint of separation of the unhybridized probe.However, compared to hybridization in solution, the rate ofhybridization is from 7 to 10 times as slow. Lastly, in situhybridization permits the microscopic examination of DNA or RNAsequences present in cells or tissues while preserving theirlocalizations. This method is suited to cytology or histologylaboratories.

In the specific case of the detection of RNAs, the so-called “northernblot” solid-phase technique constitutes the most widely usedhybridization method, used both in medical analysis and in basicresearch. Northern blotting, while it is the only method capable ofproviding information about the size of messengers, and hence about theidentity of the target, cannot be considered for large-scale analysesbecause of its low sensitivity and difficulties of automation. Forsimilar reasons, the in situ hybridization technique likewise appears tobe unsuitable for large-scale studies of RNA expression profile.

Among the methods of detection of RNA based on hybridization insolution, the nuclease protection technique has the advantage of being avery sensitive and specific method. In this technique, single-strandedRNA or DNA radioactive probes are hybridized in solution with RNApreparations containing the target RNA. After hybridization, theunhybridized nucleic acids (both the unhybridized probe and the RNAs ofthe preparation) are degraded by the action of nucleases specific forsingle-stranded nucleic acids (in general, RNases A and T1 for RNAprobes, and nuclease S1 for RNA or DNA probes). The hybridization of theprobe with its complementary RNA target “protects” the probe fromdegradation by the nuclease, and results in double-stranded moleculeswhose length is defined by the probe-target complementarity.

On the other hand, nonspecific hybridization of the probe with RNAsother than the target results in double-stranded molecules which areshorter than those originating from the specific hybridization. The twotypes of products, specific and nonspecific, may hence be distinguishedaccording to size, the most usual analysis being polyacrylamide gelelectrophoresis. An alternative technique of analysis by chromatographicseparation has been proposed in WO 95/113116.

While the nuclease protection technique has the advantage of beingsensitive and specific, the analytical method consisting of anelectrophoretic or chromatographic separation is incompatible withautomation of the method and use of a large number of samples.

The present invention is designed to permit specific detection ofnucleic acids, in particular of RNA, in a format which makes it possibleto handle simultaneously a large number of samples by a procedure whichcan be fully automated, while being sensitive, specific a reproducible.

Another object of the present invention is, in particular, to provide amethod which permits the specific detection of mRNAs from total RNAs.

The invention is based on the use of a method of hybridization insolution of nucleic acids with a nucleotide probe which, in addition toa specificity of recognition brought about by its sequence, possessesmodifications which enable it to accomplish two different functions. Thefirst function permits binding to a solid support of the probe-targethybrid, or of the probe originating from a previously denaturedprobe-target hybrid. The second function of the probe permits specificdetection by radioactive or cold detection methods.

Generally speaking, the invention involves a methodology comprising:

(1) a hybridization in the liquid phase using a doubling labeled nucleicacid probe that hybridizes specifically with a given RNA or DNA.

(2) a degradation by nucleases of the unhybridized nucleotide sequences.

(3) separation of the hybrids by specific capture on a solid support ofthe hybrids formed, by means of a modification of the probe.

(4) detection of the hybrids as a result of the abovementionedmodification, or by means of another modification present on the probe.

More specifically, the subject of the present invention is a method fordetecting nucleic acids, characterized in that it entails the followingsteps:

a) hybridization is carried out in solution of a large nucleic acidprobe, in particular one having more than 100 nucleotides, capable ofhybridizing with a given RNA or DNA in a sample containing nucleicacids, said probe comprising detection components and first capturecomponents on a solid support;

b) degradation of the unhybridized nucleotide sequences in the sample iscarried out with an enzyme that degrades unhybridized nucleic acids,

c) capture of the hybrids obtained in step b) is accomplished bybringing them into contact with a solid support coated with the secondcapture components that interact with said first capture components ofsaid probe, and

d) detection of the hybrids, or of the probes originating from thehybrids after denaturation, is accomplished by means of said detectioncomponents.

The use of large probes, in particular ones having more than loonucleotides, confers greater specificity of hybridization and thepossibility of introducing sufficient modifications for the detectionand capture of the probe while preserving the stability of thehybridization.

In particular, the probe contains from 100 to 1000 nucleotides,preferably from 250 to 500 nucleotides.

One aspect of the invention is associated with the use, after incubationwith nucleases, of one or more techniques of removal of oligonucleotides(less than 100 bases), such as differential precipitation, filtrationthrough membranes, gel filtration (exclusion chromatography) or anyother technique permitting separation of the target-probe hybrids fromthe degradation products of nucleic acids, in particular of theunhybridized probe.

In general the two functions of the probe are accomplished by twodifferent nucleotide modifications. However, it should be noted that thesame modification may be used for capture and for detection.

According to the present invention the probe comprises a sufficientamount of detection components and of capture components to effectcapture and then a sufficient detection. However, the amount of saiddetection components and of said first capture components bound to theprobe is optimized in order to be compatible with the hybridization ofstep a) and the nondegradation of the hybrids in step b).

It should be borne in mind that the introduction of modified nucleotidesinto the bifunctional probes can cause a destabilization of the specifichybrids in respect of the pairings close to the modifications. Itemerges, in effect, from the examples illustrating the invention whichare described below that, if the amount of the modifications of theprobe is too large, the hybridization is insufficiently stable, so thateven the hybrids can become degraded by the enzymes used in step b).

Preferably, when the detection components and first capture componentsare biological molecules substituted on nucleotides constituting theprobe, the latter should not contain more than 15%, preferably not morethan 10%, of nucleotides modified with said detection components andsaid first capture components. It will also be understood that theseamounts are dependent on the size of the molecules constituting thedetection components and capture components. If the steric hindrance istoo great, they will weaken the stability of the hybridization.

The nucleotide probes according to the present invention can be DNAprobes or, preferably, RNA probes. The RNA probes can be obtained by invitro transcription and possess some advantages over DNA probes. RNAprobes occur directly in the form of a single strand, so they do notrequire denaturation. Moreover, there is no interference with acomplementary strand as in the case of DNA probes. Thus, RNA probes formmore stable hybrids with their targets than those obtained with DNAprobes.

Advantageously, to detect an mRNA expressed from a given gene, an RNAprobe obtained by in vitro transcription of cloned cDNA fragments ofsaid gene is used.

Advantageously, an RNA probe obtained by in vitro transcription usingnucleotides, some of which are modified by coupling to a said detectioncomponent and/or a said capture component, is used.

Numerous modifications may be incorporated in the RNAs transcribed bythe RNA polymerases SP6, T3 and T7. For example, ribonucleotidescontaining biotin groups (such as biotin-UTP and DIG-UTP) or fluoresceingroups (such as F1-11-UTP, F1-12-UTP) exist, which can replace UTP inthe in vitro transcription reactions catalyzed by the RNA polymerasesSP6, T3 or T7. In addition, other types of modifications may beincorporated in ribonucleotides according to the techniques known to aperson skilled in the art.

After hybridization of the bifunctional probe with the preparationcontaining the target RNA, the unhybridized probe has to be degradedunder conditions which do not bring about a degradation of the specificprobe-target hybrids. The method chosen to remove the unhybridized probemust also take account of the possible existence of single-strandedregions in the probe-target hybrid, in order not to lose a portion ofthe specific signal. There are several enzymes which have the capacityto hydrolyze single-stranded nucleic acids specifically. Among others,there may be mentioned nuclease S1, mung bean nuclease or exonucleaseVII, which do not have sequence specificity, and RNase A, RNase CL3,RNase Phy M, RNase T1 or RNase U2, which cut only upstream and/ordownstream of certain nucleotides. The use of one or more nucleolyticenzymes which are specific for single-stranded regions and are notspecific for the modified nucleotides incorporated in the probe formpart of the invention. For example, if the riboprobes containmodifications linked to UTP, RNase T1 (which cuts on the 3′ side of G)and/or RNase CL3 (which cuts on the 3′ side of C) and/or RNase U2 (whichcuts on the 3′ side of A) should preferably be used so as to decreasethe probability of cutting around the modified UTPs in the probetargethybrid.

Advantageously, when mRNAs are detected, in step a) an RNA probe isused, and in step b) the enzymatic degradation is carried out with anRNase.

When the detection components and said first capture components aresubstituted on given nucleotides, it is preferable to use in step b) anenzyme which couples the single-stranded nucleic acids at a nucleotideother than said substituted nucleotides.

In particular, the detection components and said first capturecomponents are substituted on Uridine nucleotides and the enzyme of stepb) is an RNase T₁.

The capture group permits specific binding of the probe to the solidphase.

In one embodiment, said first capture components of said probe consistof a first biological molecule bound covalently to said probe and saidsecond capture components of said solid support consist of a secondbiological molecule bound to the solid support, which second biologicalmolecule interacts and binds noncovalently to said first molecule.

Special mention may be made of the embodiment in which said first andsecond molecules constitute the biotin/streptavidin system orantigen/antibody system, or still more especially the first and secondcapture components can be the system comprising digoxigenin (DIG) andanti-DIG antibody or fluorescein and an anti-fluorescein antibody; moregenerally, any ligand-receptor or hapten-antibody system.

According to the invention, the capture of the probe-target hybridnucleic acids, or of the probe originating from a previously denaturedprobe-target hybrid, takes place on a solid surface. This surface ispreferably, but is not limited to, a microplate. Capture can take placeon the surface of a plasma resonance detector (Surface plasmon resonanceFisher et al. 1994, Curr. of Biotech. 5, 389-395). The surface chosenpermits specific binding of the probe as a result of the immobilizationof the molecules having a high affinity for the capture groups presentin the probe.

The detection group permits specific demonstration of the probe. Forexample, biotin may be recognized by streptavidin or by an anti-biotinantibody coupled to an enzyme; DIG by anti-DIG antibodies coupled to anenzyme, or fluorescein by anti-fluorescein antibodies coupled to anenzyme. The detection may also be mediated by the presence of aradioactive nucleotide in the probe.

The detection component of the probe can be a component which isdirectly or indirectly detectable. In effect, “detection component” isunderstood to mean a molecule which can be detected directly orindirectly, that is to say, in the latter case, after binding byinteraction or covalent coupling with another molecule and/or a solidparticle. “Direct detection” is understood to mean, in particular, thecases where said molecule itself contains a detectable component such asa radioactive or fluorescent component, or where said molecule iscoupled to an enzyme which can be detected using a substrate oralternatively said molecule is coupled to a fluorescent molecule.“Indirect detection” is understood to mean, in particular, the caseswhere said molecule is a biological molecule capable of reactingphysico-chemically by a noncovalent interaction or by covalent couplingwith another biological molecule itself containing a directly detectablecomponent such as a radioactive or fluorescent atom, an enzyme or afluorescent molecule.

The indirectly detectable detection component can consist of abiological molecule capable of reacting noncovalently with anotherbiological molecule containing a directly detectable component such asan enzyme. The streptavidin/biotin or antigen/antibody systems may bementioned especially. In particular, the indirectly detectable detectioncomponent is chosen from biotin, fluorescein and DIG.

In the case of radioactive detection, the presence of a radioactivenucleotide in the probe can be, for example, discerned by counting in adetector of radioactivity suitable for microplates. In the case of colddetection, the detection group incorporated for this purpose in theprobe may be recognized using a specific ligand/antibody conjugated toan enzyme, followed by incubation with a substrate. Thus, biotin may berecognized by streptavidin or by an anti-biotin antibody coupled to anenzyme; DIG by anti-DIG antibodies coupled to an enzyme, or fluoresceinby anti-fluorescein antibodies coupled to an enzyme. The enzymes usedfor cold detections can be, inter alia, alkaline phosphatase, peroxidaseor b-galactosidase. There are numerous colorimetric, fluorescent orchemiluminescent substrates for these enzymes. The measurement of enzymeactivity may be carried out by automatic reading in a colorimeter,fluorimeter or luminometer suitable for microplates.

Alternatively, if the chosen capture surface is that of a plasmaresonance biosensor, the presence of detection groups in the probe maybe determined by direct measurement of the interaction with specificligands/antibodies.

When the capture and detection components are biological moleculessubstituted on a nucleotide, in particular Uridine, biotin, fluoresceinor digoxigenin may be mentioned as detection and/or capture component.More especially, the detection component is digoxigenin and/or biotinand said first capture component is chosen from biotin and digoxigenin,respectively. In one embodiment, said first capture component is biotinor digoxigenin, and nucleotides carrying these modifications areUridines and said detection component is DIG or biotin, respectively.

Preferably, the detection group is DIG, the conjugate is an anti-DIGantibody coupled to alkaline phosphatase and the substrate permits achemoluminescent detection.

In one embodiment illustrated by the examples of the detaileddescription which follows, the probe contains from 2 to 5% of thenucleotides modified by coupling to a said detection componentconsisting of a biotin molecule, in particular 3 to 4%, and from 2 to 5%of the nucleotides modified by coupling to a said first capturecomponent consisting of a digoxigenin molecule, in particular 2 to 3%.

The subject of the present invention is also a diagnostic method inwhich a detection method according to the present invention is used,comprising the detection of nucleic acids involved in a pathology.

The subject of the present invention is also a kit which is useful forcarrying out a detection or diagnostic method according to the presentinvention, characterized in that it contains large RNA probes, inparticular ones having more than 100 nucleotides, carrying detectioncomponents and said first capture components on a solid support, and asolid support coated with said second capture components.

Other features and advantages of the present invention will becomeapparent in the light of the description and the examples which follow,reference being made to FIGS. 1 to 10.

FIG. 1 A depicts the results for the binding of 1 fmol of (32aP)CTP-labeled biotin-DIG riboprobe, and enables the percentage of biotinintroduced for incorporated in the riboprobe which is necessary for goodbinding to a plate to be determined. The riboprobes studied are obtainedby reverse transcription using a variable proportion of biotin-UTP (5,10, 15, 25, 100%) and a fixed amount of DIG-UTP (10%). The detection ofthe riboprobes is performed by measurement of the radioctivity.

FIG. 1B depicts the detection of 300 amol of riboprobes using anti-DIGantibody (cold detection system). The riboprobes studied are formed froma varible percentage of biotin-UTP (5, 10, 15, 25%) and 10% of DIG-UTP.

FIG. 2A shows the binding of (α-³²P) CTP-labeled biotin-UTP (25 and100%) riboprobes with a high specific activity (2×10⁵ cpm/fmol). Thisenables the sensitivity of the binding to be determined more precisely.The riboprobes undergo doubling dilutions from 2.5 to 0.01 fmol.

FIG. 2B shows the results for the detection of biotin-DIG riboprobes asa function of the percentage of DIG in the riboprobe, using an anti-DIGantibody coupled to alkaline phosphatase followed by measurement offluorescence.

FIG. 3A depicts the capture of riboprobes (biotin-UTP) hybridized withan (α-³²P) CTP-labeled complementary nucleotide sequence. This enablesthe binding of the riboprobes in double-stranded form to be studied. Theriboprobes studied are prepared from a variable percentage of biotin-UTP(5, 15, 25, 35%). The hybrids are subjected to doubling dilutions from 2to 0.015 fmol. The detection of the hybrids is performed by measurementof the radioactivity. These curves show the optimum amount of biotin-UTPin the riboprobe (in double-stranded form) for obtaining good binding.

FIG. 3B depicts the capture of riboprobes (5, 15, 25, 35% biotin-UTP)hybridized with a (α-³²P) CTP-labeled nucleotide sequence (35% DIG-UTP).This enables the binding of the riboprobes in double-stranded form to bestudied. The detection of the hybrids is performed by measurement of theradioactivity.

FIG. 4 shows the influence of the percentage of DIG-UTP on the size ofthe detection signal using anti-DIG antibodies. The probes studiedconsist of 15% biotin-UTP and (5, 15, 20, 25% of DIG-UTP)

FIG. 5 shows the use of DIG-UTP riboprobes in a functional test ofnuclease protection.

The riboprobes studied consist of DIG-UTP (0, 5, 15, 25 and 35%). Theyare radiolabeled with (α-³²P) CTP.

Each riboprobe is hybridized with a complementary ribonucleotidesequence (10 or 1 ng) in the presence of 10 μg of yeast RNA (+). Acontrol without digestion by Rnase T1 (−) and an RNase control without acomplementary probe (0) were performed for each probes.

FIG. 6 depicts the autoradiograph of a polyacrylamide gel, and shows thesensitivity of detection using a radiolabeled riboprobe consisting of15% of biotin-UTP and 10% of DIG-UTP compared to the same radiolabeledriboprobe unmodified.

The two radiolabeled riboprobes used at 1 fmol (45,000 cpm) were placedwith a decreasing amount of a cold complementary ribonucleotide sequence(subjected to tenfold serial dilutions from 10⁴ to 10³ pg) in thepresence of 10 μg of yeast RNA (+). A probe control without digestion byRnase T1 (−) and an RNase control without a complementary probe (0) wereperformed for each probes. The band visible on the gel corresponds tothe riboprobe which has not been degraded (since in hybrid form) on thedigestion with Rnase T1.

FIG. 7 depicts the autoradiograph of a polyacrylamide gel. It shows thedetection of the messenger of the β-actin gene in different human or ratcells using a riboprobe (complementary to a 250 nucleotides of themessenger of the mouse β-actin gene) consisting of 15% of biotin-UTP and10% of DIG-UTP and radiolabeled with (α-³²P) CTP. The order of thedifferent cells or tissues tested is: brain (rat), kidney (rat), liver(rat), Jurkat (human), HepG2 (human), H4II (rat), placenta (human),yeast, Hela (human). For each sample, a control of the riboprobe withoutRnase T1 (−) and a detection of the mRNA of the β-actin gene (+) werecarried out. Control of the specificity of the reaction is carried outusing yeast.

FIG. 8 depicts a detection on plates (after protection from digestion byribonuclease T1) of the β-actin messenger with a riboprobe consisting of15% of biotin-UTP and 10% of DIG-UTP, radiolabeled with (α-³²p) CTP(3×10⁴ cmp/fmol).

The detection of the presence of the riboprobe is carried out aftercutting out the wells and counting the radioactivity on a BeckmanLS6000IC counter. The β-actin mRNA is tested for in 10 μg, 5 μg and 1 μgof total RNAs extracted from different tissues (see legend).

FIG. 9 depicts a detection on plates of an uncommon messenger, that ofHNF1 (hepatic nuclear factor 1) using a riboprobe (15% biotin-UTP, 10%DIG-UTP) radiolabeled with α-³²P-CTP (5×10⁴ cpm/fmol). The samplestested correspond to 10, 5 and 1 μg of tRNA originating from kidney,liver, placenta and yeast.

FIG. 10 depicts a detection on plates of the β-actin messenger with ariboprobe consisting of 15% of biotin-UTP and 10% of DIG-UTP. Thedetection of the riboprobe is carried out using an anti-DIG antibodycoupled to alkaline phosphatase. The measurement is carried out byluminescence.

In the examples, modified riboprobes obtained by transcription fromnucleotide monomers designated NTP, comprising modified nucleotidesUTP-biotin (Boehringer, Ref. 1388908) andUTP-DIG (Boehringer, Ref.1209256), are used.

When it is stated that riboprobe is obtained from n% of biotin-UTP orDIG-UTP, this means that the percentage of biotin-UTP relative to thetotal amount of modified and unmodified UTP used for the transcriptionis n%. This corresponds to a percentage of corresponding modifiednucleotide in the riboprobe obtained of approximately n/4%.

Example 1 shows the bifunctional aspect of the biotin-UTP and DIG-UTPriboprobes. Example 2 underlines the importance of the degree ofmodification in relation to the test of protection from digestion byribonucleases RPA. Example 3 illustrates an application of the inventionas a method for the detection of gene expression.

EXAMPLE 1 DEMONSTRATION OF THE CAPTURE AND DETECTION OF A RIBOPROBECONSISTING OF BIOTIN-UTP AND DIG-UTP ON A MICROPLATE

With the projects relating to the sequencing of the human genome, aconsiderable effort has been expended in relation to the automation ofDNA sequencing methods. However, the traditional methods of analysis ofgene expression are not suited to the detection of an ever increasingnumber of messengers. Thus, a major advantage of a new method fordetecting messenger RNAs lies in its potential for automation. And mustbe able to be carried out on a solid support such as a microplate, whichis especially suitable for studying large series of samples. The use ofa riboprobe possessing a functional portion permitting, on the one handits capture, and possessing a property of specific recognition is seento be very suitable for the detection of messenger RNAs. In the examplepresented, the capture is effected by biotin and the detection by DIG.

1. General Principle of the use of a Riboprobe.

A DNA segment corresponding to a portion of a gene is inserted at acloning site immediately downstream of a bacteriophage (T3, T7 or SP6),RNA polymerase promoter, in an orientation which leads to the productionof an antisense RNA. The recombinant plasmid is then cut with arestriction enzyme on the 3′ side of the insert. The linearized plasmidis then transcribed in the presence of ribonucleotides comprisingmodified ribonucleotides, for example biotin-UTP and DIG-UTP. An excessof this doubly labeled RNA is hybridized in solution with the mRNAs tobe tested. The hybridizations are carried out under standard stringentconditions at T=40-50° C., overnight (16 hours) in an 80% formamide, 0.4M NaCl buffer at a pH of between 7 and 8. The unhybridized probe is theneliminated by digestion using ribonucleases specific for single-strandedRNA, such as the RNases CL3, T1, Phy M, U2 or A. The presence of thefirst modification, for example biotin-UTP, in the mRNA:riboprobe(biotin-DIG) hybrid enables it to be captured on a microtitration platewhose surface is coated with streptavidin. The presence of anothermodification, for example DIG, enables the hybrid to be detected andquantified by an ELISA method using an anti-DIG antibody coupled toalkaline phosphatase. Moreover, it is also possible to carry out atranscription in the presence of (α-³²P) CTP in order to be able todetect and quantify the hybrid (mRNA:riboprobe) after application to aplate.

2. Model System: HNF1 (hepatic nuclear factor 1)

A 490 bp PvuII fragment of a cDNA coding for rat HNF1 (992 to 1482,Chouard et al. 1990, NAR 18, 5853-5863) is subcloned at the SmaI site ofthe vector pBS (Stratagene). The antisense riboprobe is obtained afterlinearization of the vector by cleavage with EcoR1, followed by atranscription carried out with T3 RNA polymerase. Synthesis of the senseriboprobe is accomplished after cleavage of the vector with HindIII,followed by a transcription with T7 RNA polymerase.

3. Synthesis of the Riboprobe

Each transcription reaction with T7 or T3 RNA polymerase is carried outin an Eppendorf tube with the following conditions: DEPC H20 q.s. 20 μl;40 mM Tris-HCl pH 7.5; 6 mM MgCl2; 2 mM spermidine; 5 mM NaCl; 10 mMDTT; 100 μg/ml bovine serum albumin (BSA) (fraction V, Sigma); 500 μMCTP, ATP, GTP; variable amounts of UTP, biotin-UTP and DIG-UTP,depending on the desired percentage of modification (for example, toobtain a 15% biotin and 10% DIG riboprobe: 75 μM biotin-UTP; 50 μMDIG-UTP, 375 μM UTP); 1 U/ml of ribonuclease inhibitor; 40 U of T7 or T3RNA polymerase; 1 μg of plasmid DNA linearized after digestion with asuitable restriction endonuclease (EcoR1 or Hind III). The mixture isincubated for 2 hours at 37° C. When transcription is complete, theplasmid DNA is digested by adding 1 μl (1 unit) of DNase RQ1 (Promega).The reaction is incubated for 15 minutes at 37° C. The unincorporatedribonucleotides are removed by passage through a Biospin 30 exclusionchromatography column (Biorad).

In order to quantify the synthesis of the riboprobe or determine thepresence of a hybrid (mRNA:riboprobe) radioactively, the transcriptionis carried out with 1-7 μl of (a32P)CTP (800 Ci/ml; 20 mCi/ml),depending on the desired specific activity. The quality of the riboprobeis determined by migration on a 5% acrylamide electrophoresis gel underdenaturing conditions.

4. Capture of the Riboprobes on Microplates

Microplate wells linked to streptavidin (Boehringer 1602853) are coveredwith 100 μl of hybrid diluted in 1×PBS buffer; 1% BSA (bovine serumalbumin) for 1 hour at 37° C. The plates are then washed 5 times withwashing buffer: 0.1 M NaCl; 0.1 M Tris-HCl; 0.1% Tween 20; 3 mM MgCl2,pH 7.5. 100 μl (70 mU) of anti-DIG antibody coupled to alkalinephosphatase (Boehringer) are then added and incubated for 1 hour at 37°C. in washing buffer containing 1% BSA.

5. Detection of the Riboprobes on Microplates

Depending on the modification of the probe, the detection can be cold(fluorescent or luminescent) or radioactive.

5.1. Fluorescent Detection

The reaction is carried out with 100 μl of 4-methylumbelliferylphosphate (Sigma) substrate at a concentration of 0.4 mg/ml in 0.1 Mdiethanolamine buffer, pH 9.8. Incubation is carried out overnight atroom temperature. Measurement of the fluorescence is carried out with afluorimeter (Dynatech) with an excitation wavelength of 365 nm and anemission wavelength of 450 nm.

5.2. Luminescent Detection

The reaction is carried out with 100 μl of AMPPD substrate (Tropix)diluted to {fraction (1/59)} in the presence of Sapphire enhancer(Tropix) diluted to {fraction (1/10)} in 0.1 M diethanolamine buffer, pH9.8. The measurement of the luminescence is carried out after 20 minutesat room temperature on a Micro Beta Trilux luminometer (Wallac).

5.3 Radioactive Detection

This involves the use of radiolabeled riboprobes. After being cut out,each well is brought into contact with 3 ml of scintillation fluid. Themeasurement of the radioactivity is carried out on a Beckman LS6000ICcounter.

6. Results

Biotin-UTP was incorporated in the riboprobe, which enables it to becaptured specifically by streptavidin previously immobilized on amicroplate.

For all the plate binding studies a radiolabeled riboprobe notpossessing biotin-UTP in its sequence was used as a control ofspecificity.

6.1 Influence of the Percentage of Biotin on the Degree of Capture of aSingle-stranded Riboprobe.

As shown in FIG. 1A, the capture of the single-stranded riboprobe(biotin-UTP, (a32P) CTP) is correlated with the amount of biotinincorporated in the riboprobe. The smallest binding was obtained with 5%of biotin. Then increases linearly up to 25% of biotin, to reach aplateau. The detection is carried out by measurement of theradioactivity. When a riboprobe containing 100% of biotin-UTP is used,the maximum degree of capture is 80%.

The influence of the percentage of biotin-UTP on the degree of capturemay also be discerned with a cold detection system. However, it isnecessary to incorporate DIG-UTP in the riboprobe. FIG. 1B shows such adetection system. In agreement with the above results, the degree ofcapture increases with the percentage of biotin-UTP.

6.2. Threshold of Sensitivity of the Different Detection Systems.

Different types of detection may be used, radioactive or cold.

Radioactive detection is carried out after the incorporation of aribonucleotide radiolabeled with (a32P) UTP or (A32P) CTP.

Cold detection is possible as a result of the incorporation of amodified nucleotide such as biotin, digoxigenin or fluorescein, detectedwith a conjugate consisting of a specific ligand (streptavidin forbiotin) or a specific antibody (anti-biotin, anti-DIG, anti-fluorescein)coupled with an enzyme (alkaline phosphatase, peroxidase). Visualizationis carried out by measuring the appearance of a fluorescent orluminescent product.

Hereinafter, the detection is carried out either by the incorporation ofa radioactive nucleotide (A32P) CTP, or by the incorporation of DIG-UTPfollowed by a measurement of fluorescence or of chemiluminescence.

In the case of radioactive detection, to obtain the greatest possiblesensitivity, riboprobes having a high specific activity (2×105 cpm/fmol)were used. Under these conditions, FIG. 3A shows that it is possible todetect up to 1 attamole of riboprobe on the plate.

For cold detection, as in radioactive detection, the threshold ofsensitivity depends on the level of incorporation of the modifiednucleotide used for detection (DIG-UTP).

For example, FIG. 2B shows that the intensity of the signal increaseswith the percentage of DIG-UTP present in the riboprobe: a probeconsisting of 25% of DIG-UTP is detected with 2.5 times the intensity ofa probe having 5% DIG-UTP. Under conditions of incorporation of DIG-UTPcorresponding to the detection maximum (35% DIG-UTP), the threshold ofdetection is 10 attamoles in fluorescence and 5 attamoles inluminescence.

These results show that it is possible to detect with different systems(radioactive or cold) riboprobes captured on microplates. In all cases,the threshold of detection depends on the level of modification of theprobe.

6.3 Capture and Detection of Double-stranded RNA Molecules.

After it was verified that it was possible to capture and detect asingle-stranded RNA molecule on a microplate, the possibility of captureof double-stranded molecules was studied. In actual fact, the presenceof a given mRNA in a sample is verified by hybridization with ariboprobe. The hybrid formed is protected from degradation byribonucleases, whereas the single-stranded molecules are removed bydigestion. The detection of hybrid molecules is hence the feature whichdiscloses the presence of an mRNA. The double-stranded molecules studiedcontain a different percentage of modifications.

FIG. 3A shows the study of the binding of the riboprobes (5, 15, 25, 35%biotin-UTP) after formation of a hybrid with an (α-³²P) CTP-radiolabeled complementary ribonucleotide sequence. It should be noted that,in order to be able to monitor the behavior of the hybrid on plates,only the complementary strand is radiolabeled.

It is apparent that the optimum binding of the hybrid occurs at between15 and 25% of biotin-UTP.

As for the single-stranded riboprobe, 5% of biotin-UTP is insufficientto bind the hybrid properly.

FIG. 3B shows the influence of the degree of biotin-UTP on the captureof hybrid consisting of (5, 15, 25, 35%) biotin-UTP and 35% DIG-UTP.

The maximum capture of the biotin-UTP, DIG-UTP hybrid is obtained with15% of biotin-UTP.

These results confirm those obtained above. Moreover, a probe containing35% of biotin-UTP gives rise to a very low level of binding of thehybrid. Probably as a result of a lower stability of the duplex obtainedin the presence of 35% DIG-UTP. This result demonstrates that anexcessive modification of the riboprobe destabilizes the hybrid, andmanifests itself in a very low level of capture.

It is also possible to detect double-stranded RNA molecules usinganti-DIG antibodies coupled to alkaline phosphatase, followed by ameasurement of fluorescence (results not shown).

For example, FIG. 4 demonstrates that optimum detection of the hybrid isachieved when the riboprobe containing 15% of biotin-UTP also contains15% of DIG-UTP.

These results collectively indicate very good binding of the riboprobecontaining 15% of biotin-UTP, in both single-stranded anddouble-stranded form.

The results obtained show the potential possessed by the biotin-UTP,DIG-UTP riboprobes (in single and double-stranded form) for binding toand being detected on microplates. These riboprobes possessing bothcapture and detection properties are seen to be very suited to amessenger RNA detection which can be automated. However, differentplate-binding behavior between a single and double-stranded molecule wasobserved. For a single-stranded molecule, the degree of capture islinked directly to the amount of biotin-UTP incorporated in theriboprobe. Whereas for a double-stranded molecule, the degree of capturepasses through an optimum which is reached when the riboprobe consistsof 15% of biotin-UTP. Thus, for double-stranded molecules, the degree ofbinding is not directly correlated with the amount of biotin containedin the hybrid, but is a consequence of the stability of the hybrid andpercentage of modification. This implies the need to perform anoptimization of the binding of double-stranded molecules.

EXAMPLE 2 EFFECT OF THE INCORPORATION OF MODIFIED NUCLEODIDES IN ARIBOPROBE ON THE STABILITY TO DEGRADATION BY RIBONUCLEASES SPECIFIC FORSINGLE STRANDS

Example 1 shows that an excessive proportion of modification bringsabout a destabilization of double-stranded RNAs. It is hence essentialto determine the consequences of such modifications in relation to thetest of protection from digestion with ribonucleases. Moreover, in thistest, the pairing of the two strands of the hybrid has to be verystable, since any unpairing gives rise to a cleavage by theribonucleases and a loss of the specific signal. On the other hand,since the modifications are located on the uridine, it is probable thatthe ribonucleases which cut at this nucleotide are less tolerant thanthose which cut after another nucleotide.

Different types of ribonuclease are available on the market (C13, T1, A,PhyM, U2). They have the common feature of only degrading an RNAmolecule in single-stranded form and of leaving double-strandedmolecules intact. These enzymes differ in the specificity of theircleavage sites.

Traditionally, the ribonuclease protection test is carried out.with amixture of ribonucleases A and T1. However, it was verified thatribonuclease A, which cuts after uridine, cytidine and thymidineresidues, is less usable than ribonuclease T1 which cuts only afterguanosine (not shown). This made it possible to eliminate ribonuclease Afrom the test, and to retain only ribonuclease T1 which affords a bettercompromise between the preservation of the specific signal(double-stranded molecule) and the degradation of the nonspecific(single-stranded) signal. In order to avoid the presence of a highbackground during visualization on microplates, it is necessary for theunhybridized probe to be completely digested.

1. Synthesis of the Probes

The synthesis of the HNF1 probes and antiprobes was carried out as forExample 1.

2. Hybridization

One fmol of HNF1 antisense probe containing different modifications(biotin and/or DIG) is hybridized with 10 and 1 ng of sensecomplementary sequences in the presence of 10 μg of yeast t RNA. Thehybridization buffer consists of 40 mM PIPES pH 6.4; 1 mM EDTA pH 8.0;0.4 M NaCl; 80% deionized formamide. After denaturation of the mixtureat 93° C. for 4 minutes, hybridization is carried out aat 43° C. for 16hours.

3. Protection From Digestion by RNase T1

Digestion with RNase T1 is carried out after hybridization in order todegrade the unhybridized riboprobe and mRNAs.

Digestion with RNase T1 is carried out by adding 300 μl of digestionbuffer: 300 mM NaCl; 10 mM Tris-HCl pH 7.4; 5 mM EDTA pH 7.5; 20 U RNaseT1 (Boehringer). Digestion of the unhybridized RNAs is carried out at37° C. for 30 minutes. The RNase T1 is then inactivated using 20μl of10% SDS solution and 10 μl of a proteinase K solution at a concentrationof 10 mg/ml. The mixture is incubated for 30 minutes at 37° C. Thehybrids are extracted with 400 μl of phenol/chloroform/isoamyl alcoholmixture (Amesco). After centrifugation at 12,000 rpm for 5 minutes, theupper phase is transferred to a fresh tube. The hybrids are precipitatedin the presence of 200 μl of 4M ammonium acetate and 750 μl of absoluteethanol for 30 minutes at −20° C.

Depending on the protocol for analysis of the hybrids, the pellet istaken up:

either in 10 μl of loading buffer comprising 80% formamide; 0.1% xylenecyanol; 0.1% bromophenol blue; 2 mM EDTA. The pellet is then analyzed onacrylamide gel under denaturing conditions,

or in 100 μl of capture buffer: 1×PBS; 1% bovine serum albumin. Thesample is subsequently analyzed after binding to a microplate coatedwith streptavidin (Boehringer).

4. Analysis on Polyacrylamide Electrophoresis Gel

After denaturation of the sample by heating to 93° C. for 4 minutes, itis subjected to electrophoretic migration at 40-45 V/cm in a denaturinggel containing 5% of polyacrylamide; 7M urea containing 1×TBE.

The radioactivity is detected by autoradiography. The size of the bandcorresponding to the riboprobe is determined by comparison with themigration of radiolabeled DNA fragments of known sizes.

5. Results

The tests were directed towards determining whether the capture (biotin)or detection (DIG) modifications of the riboprobe are compatible withthe nuclease protection tests. The test used is a protection fromdigestion by RNase T₁, followed by a traditional analysis onelectrophoresis gel under denaturing conditions. The size of the HNF1riboprobe is 500 nucleotides.

Biotin-UTP (35% and 100%) riboprobes (results not shown) and DIG-UTP (5,15, 20 and 35%) riboprobes (results depicted in FIG. 5) were tested.

It is apparent that the higher the percentage of modification, the lessthe riboprobe is usable in the ribonuclease protection test. Thus, it isnot possible to use riboprobes consisting of 35% of biotin-UTP and 35%DIG-UTP since there is a complete loss of the specific signal due todegradation by the ribonucleases, probably as a result of the strains towhich these modifications give rise in the hybrid. Likewise, 100% of UTPmodification is not tolerated, irrespective of the modification(biotin-UTP, DIG-UTP).

Fine analysis of the degradation of the hybrid as a function of thepercentage of DIG-UTP incorporated in the riboprobe discloses that anincrease in the degree of modification gives rise to a progressive lossof sensitivity. The maximum threshold of DIG-UTP modification withoutsignificant loss of specific signal is obtained with a probe containing10% of DIG-UTP (FIG. 5).

FIG. 6 shows a comparative study of sensitivity between a riboprobe (15%biotin-UTP, 10% DIG-UTP) and the same riboprobe unmodified. The testused is a protection from digestion by Rnase T1, followed by atraditional analysis on electrophoresis gel under denaturing conditions.In this experiment, both radiolabeled riboprobes possess a specificactivity of 4×10⁴ cpm per fmol. The riboprobe (15% biotin-UTP, 10%DIG-UTP) is capable, like the unmodified probe, of detecting 100 pg ofcomplementary sequence, but with a weaker signal.

These results show that, in the case of the use of biotin as capturemodification and DIG in detection, 15% of biotin and 10% of DIG appearto be suitable for a good detection of the target RNAs. With othermodifications, it will be necessary to carry out an optimization.

EXAMPLE 3 USE OF A RIBOPROBE (15% BIOTIN-UTP, 10% DIG-UTP) FOR DETECTINGTHE MESSENGER OF THE b-ACTIN OR HNF1 GENE ON MICROPLATES

The synthesis of the HNF1 riboprobe, capture and detection were carriedas for Example 1.

1. Synthesis of the β-actin Probe

A 250-bp fragment of a cDNA coding for mouse β-actin (582-831) is clonedat the KpnI/XbaI site of the vector pBS (Stratagene). The β-actinriboprobe is obtained after linearization of the vector by cleavage withHindIII followed by a transcription with T7 RNA polymerase.

2. Extraction of the Total RNAs

The total RNAs are isolated from 10⁷ cells or from 10 to 100 mg oftissues using 2 ml of RNAzol B (Biotex). The tissue has to be plungedfrozen into the RNAzol B, and homogenization is carried out in a “waringblender” mill for 30 to 60 seconds before the tissue thaws, otherwisethe RNAs will be degraded by the Rnases. 0.2 ml of chloroform is thenadded to the homogenate. After being mixed and left standing for 5minutes in ice, the solution is centrifuged at 12,000 g for 15 minutesat 4° C. After the aqueous phase has been removed, an equal volume ofisopropanol is added. The mixture is allowed to precipitated at −20° C.for 30 minutes. After centrifugation at 12,000 g for 15 minutes at 4°C., the pellet is washed with 70% alcohol. The total RNAs areresuspended in DEPC water. The concentration of the sample is determinedby measuring the optical density at 280 nm. The quality of thepreparation is verified on 0.8% agarose gel.

3. Hybridization

The amount of total RNAs needed for the hybridization reaction dependson the concentration of the nucleotide sequence which is sought and thespecific activity of the radiolabeled riboprobe. With a riboprobe havinga high specific activity (>10⁹ cpm/μg), 10 μg of total RNAs are usuallysufficient to permit the detection of mRNAs which are present at a levelof 1 to 5 copies per cell.

The total RNAs (10 μg) to be analyzed are precipitated by adding 0.1volume of 3 M sodium acetate solution ph 5.2 and 2.5 volumes of coldethanol. The solution is left for 30 minutes at −20° C. The tRNAs arerecovered by centrifugation at 12,000 rpm for 15 minutes at 4° C. Thepellet is then washed with 70% ethanol solution and recentrifuged. Afterremoval of the ethanol, the pellet is dried. The tRNAs are thendissolved in 20 ml of hybridization buffer containing: 40 mM PIPES pH6.4; 1 mM EDTA pH 8.0; 0.4 M NaCl; 80% deionized formamide. 1 fmol ofthe biotin-DIG riboprobe, radiolabeled or otherwise, is then added. Thesolution is pipetted several times to permit complete solubilization ofthe pellet. The mixture is denatured by incubation at 93° C. for 4minutes. The tubes are then transferred rapidly to a water bath adjustedto the hybridization temperature. In most cases, satisfactory resultsare obtained when the RNAs are hybridized at between 45 and 50° C. for16 hours.

4. Results

FIG. 7 shows the use of a riboprobe (15% biotin-UTP, 10% DIG-UTP,(α-³²P) CTP) corresponding to a sequence complementary to the mouseβ-actin messenger. The size of the riboprobe is 250 base pairs.

The riboprobe (15% biotin-UTP, 10% DIG-UTP, (α-³²P) CTP was capable ofdetecting the β-actin messenger in 10 μg of total RNAs. The test used isa protection from digestion by Rnase T1 followed by analysis byacrylamide gel electrophoresis under denaturing conditions. The sizeintegrity of the ribonprobe is determined as a result of the presence ofradiolabeled markers.

The riboprobe is seen to be protected in all the samples tested (seearrow in FIG. 7). However, for the human samples, the presence of asecond band of smaller size (arrow), which is due to the existence of adifference in the sequence of the b-actin gene between man and mouse inrespect of an Rnase T1 cleavage site, should be noted. The β-actinmessenger was visualized in all the cells tested. The specificity of thedetection is given by yeast, which does not express β-actin.

This demonstrates that the riboprobe (15% biotin-UTP, 10% DIG-UTP) pairscorrectly with a complementary sequence located in an mRNA. And that itcan hence be used in the functional tests of protection from digestionby RNase T1.

FIG. 8 shows the results obtained after protection from digestion byRnase T1 followed by detection of the radioactivity after capture onmicroplates. The same amount of β-actin riboprobe (15% biotin-UTP, 10%DIG-UTP), 1 fmol (4×10⁴ cpm), was used for the detection on gel and onplates. As in the analysis using polyacrylamide electrophoresis gel, theriboprobe (15% biotin-UTP, 10% DIG-UTP, (α-³²p) CTP is capable ofdetecting the presence of the β-actin messenger starting from 1 μg oftotal RNAs. The yeast used as negative reaction control gives a very lowbackground (250 cpm) , compared to the 1800-4000 cpm obtained with 5 and10 μg of tRNA from cells expressing β-actin. Although much weaker(400-680 cpm), the signal remains positive with 1 μg of tRNA.

FIG. 9 shows the results obtained with a riboprobe (500 nucleotides)directed against a weakly expressed messenger, that of HNF1. The probeused, containing 15% of biotin-UTP and 10% of DIG-UTP, is radiolabeledwith (α-³²P) CTP (5×105⁵ cpm/fmol). The measurement of the radioactivityassociated with the two cells that express HNF1 (kidney and liver) issignificantly higher than that associated with the samples which do notexpress HNF1 (placenta and yeast). This result demonstrates thepossibility of detection by a riboprobe (biotin-UTP, DIG-UTP) of aweakly represented mRNA.

However, in order to be completely amenable to automation, this methodof analysis on microplates has to dispense with the use ofradioactivity.

FIG. 10 shows the results obtained with a β-actin riboprobe (15%biotin-UTP, 10% DIG-UTP), followed by detection on microplates with ananti-DIG antibody and a reading by luminescence.

The cells tested are: Jurkat, placenta, brain, yeast.

From Jurkat cells, it was possible to detect the β-actin messenger in0.5 μg of tRNA. For placenta and brain, the β-actin messenger wasdemonstrated in 2 μg of tRNA. These results indicate the very goodsensitivity of the detection method.

As when using radioactivity, the riboprobe is capable of detecting theβ-actin messenger starting from 10, 5, 2 or 0.5 μg of total RNAs.

Similar results are obtained with fluorescent detection (not shown).

These studies collectively show that the riboprobes (biotin-UTP,DIG-UTP), while retaining their specificity of recognition via theirribonucleotide sequences, are capable of binding to plates and beingdetected specifically. Their use in nuclease protection testsnecessitated an adjustment of the degree to which they were modified.

Although the nuclease protection technique is frequently used in mRNAdetection and quantification studies. It nonetheless remains unsuitablefor the analysis of large series of samples. Since the products ofnuclease protection experiments are usually analyzed on polyacrylamidegel under denaturing conditions. The possibility of detecting messengerRNAs on microplates using a riboprobe (biotin-UTP, DIG-UTP) constitutesa tool of very great analytical power, and makes the nuclease protectiontechnique applicable to the study of large numbers.

What is claimed is:
 1. A method for detecting nucleic acids comprisingthe following steps: a) performing a hybridization reaction in solutionusing a nucleic acid probe capable of hybridizing with a given RNA orDNA in a sample containing nucleic acids, said probe comprisingdetection components and first capture components; b) degrading theunhybridized nucleotide sequences in the sample with an enzyme thatdegrades unhybridized nucleic acids; c) capturing hybrids comprisingsaid nucleic acid probe and said RNA or DNA by bringing them intocontact with a solid support comprising second capture components thatinteract with said first capture components of said probe; d) detectingthe hybrids, or the probes originating from the hybrids afterdenaturation, by means of said detection components.
 2. The method ofclaim 1, wherein said nucleic acid probe is an RNA probe and wherein theenzymatic degradation is carried out with an RNase.
 3. The method ofclaim 1, wherein said first capture components of said probe comprise afirst biological molecule bound covalently to said probe and said secondcapture components of said solid support comprise a second biologicalmolecule bound to the solid support, which second biological moleculeinteracts and binds noncovalently to said first molecule.
 4. The methodof claim 1 wherein said RNA or DNA comprises an mRNA and said probecomprises an RNA probe obtained by in vitro transcription of cloned cDNAfragments, said cDNA fragments being derived from said mRNA.
 5. Themethod of claim 1, wherein said probe is from 100 to 1000 nucleotides inlength.
 6. The method of claim 1, wherein after step b), the degradationproducts of the unhybridized nucleic acids are removed.
 7. The method ofclaim 1, wherein said probe does not contain more than 15% ofnucleotides modified with said detection components and wherein saidfirst capture components comprise biological molecules.
 8. The method ofclaim 3, wherein said first and second capture molecules comprise abiotin/streptavidin system.
 9. The method of claim 3, wherein said firstcapture molecule is digoxigenin (DIG) and said second capture moleculeis an anti-digoxigenin antibody.
 10. The method of claim 1, wherein saiddetection component is a directly detectable radioactive or fluorescentcomponent.
 11. The method of claim 1, wherein said detection componentis an indirectly detectable component comprising a biological moleculecapable of reacting noncovalently with another molecule containing adirectly detectable component.
 12. The method of claim 11, wherein theindirectly detectable detection component is selected from the groupconsisting of biotin, fluorescein and digoxigenin.
 13. The method ofclaim 1, wherein said first capture component is selected from the groupconsisting of biotin, fluorescein and digoxigenin.
 14. The method ofclaim 1, wherein the detection component is digoxigenin and/or biotinand said first capture component is selected from the group consistingof biotin and digoxigenin, respectively.
 15. The method of claim 14,wherein from 2 to 5% of the nucleotides in said probe are substitutedwith a biotin molecule and from 2 to 5% of the nucleotides in said probeare substituted with a digoxigenin molecule.
 16. The method of claim 1,wherein said probe is from 250 to 500 nucleotides in length.
 17. Themethod of claim 1, wherein said probe does not contain more than 10% ofnucleotides modified with said detection components and wherein saidfirst capture components comprise biological molecules.
 18. The methodof claim 14, wherein from 3 to 4% of the nucleotides in said probe aresubstituted with a biotin molecule and wherein from 2 to 3% of thenucleotides in said probe are substituted with a digoxigenin molecule.19. The method of claim 4, wherein said in vitro transcription isperformed using nucleotides having said detection component and/or saidfirst capture components linked thereto.
 20. The method of claim 1,wherein said step of degrading the unhybridized nucleotide sequencescomnrises preferentially cutting the unhybridized nucleotide sequencesat nucleotides other than those which comprise said detection componentsand/or said first capture components.
 21. The method of claim 20 whereinsaid detection components and said first capture components are linkedto uridines in said probe and wherein said enzyme that degradesunhybridized nucleic acids comprises RNase T₁.
 22. The method of claim1, wherein said RNA or DNA comprises an RNA or DNA associated with apathology.
 23. The method of claim 1, wherein said hybrids or probes aredetected without performing gel electrophoresis or size exclusionchromatography.
 24. A kit comprising a nucleic acid probe comprisingdetection components and first capture components and a solid supportcomprising second capture components, said second capture componentsbeing capable of binding specifically with said first capturecomponents, wherein said first and second capture components comprise aligand-receptor or an antigen-antibody system and said detectioncomponents and said capture components are different and distinctmolecules.
 25. The kit of claim 24, wherein said probe is more than 100nucleotides in length.